The use of human adenovirus (Ad) vectors for transcriptionally targeted thyroid gene therapy was investigated, aiming to develop a novel therapy for feline hyperthyroidism. Ad5 (El, E3 deleted) vectors were constructed in which the feline thyroglobulin promoter controls expression of the reporter gene chloramphenicol acetyl transferase (CAT). In FRTL-5 cells, CAT expression from adenoviral constructs harbouring the expression cassette was much reduced compared to controls transfected with the same cassette in a plasmid backbone, despite higher transduction efficiencies when viral vectors were used. Transfections with the “shuttle” plasmids utilised to create the adenoviral vectors also resulted in reduced expression compared to controls. In both viruses and shuttle plasmids, consistently lower expression was noted when the cassette was in the left to right orientation than the right to left. These results suggest cis acting elements in the flanking adenoviral sequences may compromise the activity of the feline thyroglobulin promoter and thus make Ad5 an unsuitable vector for transcriptionally targeted gene therapy in feline hyperthyroidism.